Concentrations Of Various Cytokines Research Articles

Inflammatory Protein Signatures as Predictive Disease-Specific Markers for Non-Alcoholic Steatohepatitis (NASH).

Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic disease worldwide, consisting of a broad spectrum of diseases such as simple steatosis (NAFL), non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and hepatocellular carcinoma. Hepatic inflammation plays a key role in the pathophysiology of NAFLD. Inflammatory mediators such as cytokines and chemokines are considered as contributing factors to NAFLD development and progression. In the present study, we aimed to investigate the inflammatory protein signatures as predictive disease-specific markers for non-alcoholic fatty liver disease (NAFLD). This cross-sectional study included healthy control (n = 64), NAFL (n = 109), and NASH (n = 60) human subjects. Serum concentrations of various cytokines and chemokines were evaluated using sensitive multiplex assays. We used principal component analysis (PCoA) to reveal distinct differences in the levels of cytokines and chemokines between each of the study groups. Further, a random forest classification model was developed to identify the panel of markers that could predict diseases. The protein-protein network analysis was performed to determine the various signaling pathways associated with the disease-specific panel of markers. Serum concentrations of TNF-α, IL-1β, IL-1ra, G-CSF, PDGF-BB, MCP-1, MIP-1a, MIP-1b, RANTES, eotaxin, IL-8 and IP-10 were significantly increased in NASH group as compared to control group. Furthermore, serum concentrations of IL-9 and IL-13 were significantly lower in the NASH group, whereas IL-2 levels were significantly decreased in the NAFL group when compared to the control group. PCoA results demonstrated statistically significant differences in cytokines and chemokines between each of the study groups (PERMANOVA p = 0.001; R2 = 0.102). RANTES, IL-1ra, MIP-1b, IL-2, and G-CSF could differentiate the NAFL group from the controls; G-CSF, IL-1ra, TNF-α, RANTES, and IL-9 could differentiate the NASH group from the controls; and G-CSF, IL-9, IL-13, eotaxin, and TNF- α could differentiate the NASH group from the NAFL group. Our protein-protein network revealed that these markers are involved in cytokine-cytokine receptor interaction, Th1 and Th2 cell differentiation, TNF, chemokine, JAK/STAT, P13K/Akt, TLR, NOD-like receptor, NF-kB, and adipocytokine signaling pathways which might be responsible for disease pathogenesis.Our study findings revealed a set of distinct cytokine and chemokine markers and they might be considered as biomarkers in distinguishing NASH from NAFL. Future multicentre studies with larger sample size are recommended to determine the potential utility of these panels of markers.

Prednisone combined with Dihydroartemisinin attenuates systemic lupus erythematosus by regulating M1/M2 balance through the MAPK signaling pathway

ObjectiveDihydroartemisinin (DHA) plays a very important role in various diseases. However, the precise involvement of DHA in systemic lupus erythematosus (SLE), relation to the equilibrium between M1 and M2 cells, remains uncertain. Therefore, we aimed to investigate the role of DHA in SLE and its effect on the M1/M2 cells balance. MethodsSLE mice model was established by pristane induction. Flow cytometry was employed to measure the abundance of M1 and M2 cells within the peripheral blood of individuals diagnosed with SLE. The concentrations of various cytokines, namely TNF-α, IL-1β, IL-4, IL-6, and IL-10, within the serum of SLE patients or SLE mice were assessed via ELISA. Immunofluorescence staining was utilized to detect the deposition of IgG and complement C3 in renal tissues of the mice. We conducted immunohistochemistry analysis to assess the expression levels of Collagen-I, a collagen protein, and α-SMA, a fibrosis marker protein, in the renal tissues of mice. Hematoxylin-eosin staining, Masson’s trichrome staining, and Periodic acid Schiff staining were used to examine histological alterations. In this study, we employed qPCR and western blot techniques to assess the expression levels of key molecular markers, namely CD80 and CD86 for M1 cells, as well as CD206 and Arg-1 for M2 cells, within kidney tissue. Additionally, we investigated the involvement of the MAPK signaling pathway. The Venny 2.1 online software tool was employed to identify shared drug-disease targets, and subsequently, the Cytoscape 3.9.2 software was utilized to construct the “disease-target-ingredient” network diagram. Protein-protein interactions of the target proteins were analyzed using the String database, and the network proteins underwent enrichment analysis for Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways. ResultsThe results showed that an increase in M1 cells and a decrease in M2 cells within the peripheral blood of individuals diagnosed with SLE. Further analysis revealed that prednisone (PDN) combined with DHA can alleviate kidney damage and regulate the balance of M1 and M2 cells in both glomerular mesangial cells (GMC) and kidney. The MAPK signaling pathway was found to be involved in SLE kidney damage and M1/M2 balance in the kidney. Furthermore, PDN and/or DHA were found to inhibit the MAPK signaling pathway in GMC and kidney. ConclusionWe demonstrated that PDN combined with DHA attenuates SLE by regulating M1/M2 balance through MAPK signaling pathway. These findings propose that the combination of PDN and DHA could serve as a promising therapeutic strategy for SLE, as it has the potential to mitigate kidney damage and reinstate the equilibrium of M1 and M2 cells.

Plasma Virome of HIV-infected Subjects on Suppressive Antiretroviral Therapy Reveals Association of Differentially Abundant Viruses with Distinct T-cell Phenotypes and Inflammation.

The plasma virome represents the overall composition of viral sequences present in it. Alteration in plasma virome has been reported in treatment naïve and immunocompromised (CD4 count < 200) people with HIV (PWH). However, the effect of ART on virome composition in PWH on ART with preserved CD4 counts is poorly understood. We aimed to assess the alterations in plasma virome in PWH on ART in comparison to HIV-negative uninfected controls and to further investigate possible associations of plasma viruses with inflammation and immune dysfunction, namely, immunosenescence and immune exhaustion. Plasma viral DNA from PWH on ART and controls was used for sequencing on the Illumina Nextseq500 platform, followed by the identification of viral sequences using an automated pipeline, VIROMATCH. Multiplex cytokine assay was performed to measure the concentrations of various cytokines in plasma. Immunophenotyping was performed on PBMCs to identify T cell markers of immunosenescence and immune exhaustion. In our observational, cross-sectional pilot study, chronically infected PWH on ART had significantly different viral species compositions compared to controls. The plasma virome of PWH showed a significantly high relative abundance of species Human gammaherpesvirus 4, also known as Epstein-Barr virus (EBV). Moreover, EBV emerged as a significant viral taxon differentially enriched in PWH on ART, which further correlated positively with the exhaustion phenotype of T cells and significantly increased TNF-α in PWH on ART. Additionally, a significantly increased proportion of senescent T cells and IL-8 cytokine was detected in PWH on ART. Altered plasma virome influenced the inflammatory response and T-cell phenotype in PWH on ART.

CYSLTR1 antagonist inhibits Th17 cell differentiation by regulating the NF-κB signaling for the treatment of psoriasis.

Cysteinyl leukotriene receptor 1 (CYSLTR1) is observed to increase in psoriatic skin lesions. Montelukast, a CYSLTR1 antagonist, effectively treats inflammatory disorders, such as rheumatoid arthritis, multiple sclerosis, and atopic dermatitis. Thus, blocking CYSLTR1 may be a promising strategy for psoriasis immunotherapy. We prepared a montelukast sodium cream and solution and investigated their effects on psoriasis-like skin lesions induced by imiquimod (IMQ). After the treatment, serum, skin, and spleen samples were collected for evaluation. We treated human T helper (Th) 17 cells with montelukast in vitro to study its effect on Th17 differentiation and nuclear factor kappa-B (NF-κB) signaling. We also created a keratinocyte proliferation model induced by M5 cytokines and assessed the influence of montelukast on key psoriasis-related genes. We induced psoriasis in CYSLTR1 knockout (KO) mice using IMQ to explore the role of CYSLTR1 in psoriasis development. Montelukast sodium cream and solution effectively reduced the psoriasis area and severity index (PASI) and alleviated disease symptoms in IMQ-induced mice. Furthermore, reduced infiltration of inflammatory cells (Th1, Th17, and T follicular helper [Tfh] cells), decreased mRNA expression of cytokines in the skin (interleukin [IL]-17/F and IL-23), and lower serum concentrations of various cytokines (IL-2, IL-6, IL-13, and IL-17A/F) were observed. Montelukast cream and solution also decreased spleen size and the proportion of Th17 and Tfh cells, and significantly inhibited NF-κB signaling-related genes after application. Moreover, montelukast inhibited Th17 cell differentiation and suppressed NF-κB signaling in vitro. CYSLTR1 KO mice induced with IMQ showed improvement in PASI scores, serum IL-17A/F levels, and lower Th1 and Th17 cells in the spleen and skin compared to wild-type mice. Montelukast also suppressed the proliferation and inflammatory response of keratinocytes by regulating NF-κB signaling. Collectively, our results strongly indicate that inhibition of CYSLTR1 signaling to target the Th17 response holds significant promise as a therapeutic approach to manage psoriasis.

Changes in aqueous concentrations of various cytokines after intravitreal bevacizumab injection for chronic central serous chorioretinopathy.

This study aimed to investigate changes in cytokine levels after intravitreal bevacizumab injection in patients with chronic central serous chorioretinopathy (CSC). In a prospective interventional trial, 12 eyes of 12 patients with chronic CSC and 6 eyes of 6 patients who underwent cataract surgery were included as controls. Patients diagnosed as with CSC received a single intravitreal injection of bevacizumab (1.25 mg/0.05 mL). Aqueous humor samples were collected from the patients and controls. Best-corrected visual acuity and foveal thickness were evaluated, and aqueous samples were obtained before and 4 weeks after injection. The aqueous concentrations of interleukin (IL)-6, IL-8, interferon-induced protein (IP)-10, monocyte chemotactic protein (MCP)-1, platelet-derived growth factor (PDGF)-AA, and vascular endothelial growth factor (VEGF) were measured using a multiplex bead assay. After injection, the foveal thickness decreased significantly from 328 (210-477) to 283 (168-356) (p = 0.048), but the best-corrected visual acuity was not significantly different (p = 0.066). The aqueous levels of IL-8 increased significantly from 3.3 pg/mL (1.5-8.3) to 4.7 pg ⁄ mL (2.2-11.6) at 4 weeks after the injection (p = 0.046). The aqueous levels of VEGF decreased significantly from 31.4 pg/mL (17.0-53.3) to 15.2 pg/mL (7.7-21.5) (p < 0.01). No significant changes in IL-6, IP-10, MCP-1, and PDGF-AA levels were noted 4 weeks after injection (p = 0.455, p = 0.055, p = 0.076, and p = 0.339, respectively). In this study we found intravitreal bevacizumab injection decreased VEGF and increased IL-8 in the eyes of patients with chronic CSC. This study suggests the possibility that the pathogenesis of CSC may be related to abnormal circulation of the choroidal blood vessels through VEGF and IL-8 cytokine level changes.

Cytokine concentration in peripheral blood of patients with colorectal cancer.

The role of tumour secretory cytokines and peripheral circulatory cytokines in tumour progression has received increasing attention; however, the role of tumour-related inflammatory cytokines in colorectal cancer (CRC) remains unclear. In this study, the concentrations of various cytokines in the peripheral blood of healthy controls and patients with CRC at different stages were compared. Peripheral blood samples from 4 healthy participants and 22 colorectal cancer patients were examined. Luminex beads were used to evaluate concentration levels of 40 inflammatory cytokines in peripheral blood samples. In peripheral blood, compared with healthy controls and early stage (I + II) CRC patients, advanced CRC (III + IV) patients had increased concentrations of mononuclear/macrophage chemotactic-related proteins (CCL7, CCL8, CCL15, CCL2, and MIF), M2 polarization-related factors (IL-1β, IL-4), neutrophil chemotactic and N2 polarization-related cytokines (CXCL2, CXCL5, CXCL6, IL-8), dendritic cells (DCs) chemotactic-related proteins (CCL19, CCL20, and CCL21), Natural killer (NK) cell related cytokines (CXCL9, CXCL10), Th2 cell-related cytokines (CCL1, CCL11, CCL26), CXCL12, IL-2, CCL25, and CCL27, and decreased IFN-γ and CX3CL1 concentrations. The differential upregulation of cytokines in peripheral blood was mainly concentrated in CRC patients with distant metastasis and was related to the size of the primary tumour; however, there was no significant correlation between cytokine levels in peripheral blood and the propensity and mechanism of lymph node metastasis. Different types of immune cells may share the same chemokine receptors and can co-localise in response to the same chemokines and exert synergistic pro-tumour or anti-tumour functions in the tumour microenvironment. Chemokines and cytokines affect tumour metastasis and prognosis and may be potential targets for treatment.

Effects of Caffeine and 5-Caffeoylquinic Acid on Blood Cell In Vitro Cytokine Production in Response to Lipopolysaccharide Stimulation

Previous in vitro studies the have suggested anti-inflammatory properties of caffeine. Coffee is also rich in phenolic acids with several potential health benefits (chlorogenic acids), mainly 5-caffeoylquinic acid (5-CQA). The aim of the present study was to determine the effect of caffeine and 5-CQA, using concentrations comparable to typical human consumption, on LPS-induced cytokine production in whole blood in vitro cultures. Whole blood samples (n = 10) were stimulated for 24 h with LPS in the presence of caffeine or 5-CQA. The concentrations of various cytokines, malondialdehyde (MDA) and adenosine-3′,5′-cyclic monophosphate (cAMP) were determined in the culture media. Significant effects of caffeine were found only for the stimulated production of TNF-α (p = 0.014) and cAMP (p = 0.001). However, only the highest caffeine concentration (50 μM) induced lower TNF-α (p = 0.016) and higher cAMP (p = 0.006) levels in comparison to the control. The 5-CQA only influenced the stimulated production of IL-6 (p = 0.002), with lower values for the highest 5-CQA concentrations (2 μM: p = 0.012; 20 μM: p &lt; 0.001). Neither caffeine (p = 0.227) nor 5-CQA (p = 0.259) influenced MDA concentrations. In conclusion, both caffeine and 5-CQA exerted limited in vitro anti-inflammatory effects. For caffeine, these effects were attained with high physiological concentrations. However, for 5-CQA, supraphysiological concentrations were needed.

An epidermal keratinocyte homogenate induced type 2 and proinflammatory cytokine expression in cultured dermal cells

BackgroundThe “itch-scratch cycle” is a clinically well-known cause of exacerbation of atopic dermatitis (AD), but the underlying molecular mechanisms remain largely unknown. ObjectiveTo test our hypothesis that some molecules released from damaged epidermal keratinocytes by scratching inducetype 2 responses in intact skin dermal cells, leading to exacerbation of AD. MethodsNormal human dermal fibroblasts (NHDF) and human dermal blood microvascular endothelial cells (HMVEC-dBl) were treated with an epidermal keratinocyte homogenate (EKH). We used qPCR and ELISA, respectively, to measure the mRNA expressions and protein concentrations of various cytokines, including IL-6, IL-8, thymic stromal lymphopoietin (TSLP), and IL-33. We analyzed IL-33 protein expression in the nuclear fractions of NHDF by Western blotting. We also investigated the effects of IL-1R1 gene-silencing and several AD therapeutic drugs on EKH induction of cytokine production by the dermal cells. ResultsEKH significantly induced IL-6 and IL-8 in NHDF and HMVEC-dBl. EKH also induced type 2 cytokines, TSLP and IL-33, in NHDF. IL-1R1 gene-silencing in NHDF partially attenuated the induction. Dexamethasone (a corticosteroid) significantly inhibited, while ABT494 (a JAK1 inhibitor) partially inhibited, EKH’s induction of cytokines in fibroblasts. In contrast, ABT494 was more effective than dexamethasone in inhibiting the cytokine induction in HMVEC-dBl. ConclusionThis study showed that a homogenate of epidermal keratinocytes significantly induced AD-related cytokines in cultured dermal cells, and IL-1α, an alarmin, might be involved in that induction. Combined use of corticosteroids and JAK1 inhibitors is likely to block the itch-scratch cycle by targeting different types of dermal cells.

Association between aqueous humor cytokines and postoperative corneal endothelial cell loss after Descemet stripping automated endothelial keratoplasty

This study measured the intraoperative anterior aqueous humor concentrations of various cytokines during corneal endothelial transplantation and searched for relationships between these concentrations and postoperative corneal endothelial cell (CEC) depletion. We recruited 30 consecutive patients who underwent corneal endothelial transplantation with Descemet's stripping automated endothelial keratoplasty (DSAEK) at Tohoku University Hospital between February 2014 and July 2017. During surgery, we obtained aqueous humor samples and later measured the concentrations of 27 cytokines with a Multiplex Bead Assay (Bio-Plex Pro). We counted CECs 1, 6 and 12 months after surgery, and used Spearman's rank correlation coefficient to identify relationships between CEC depletion and the concentrations of detected cytokines. The loss of CECs 1-6 months after surgery was significantly correlated with IL-7, IP-10, MIP-1a and MIP-1b concentrations (-0.67, -0.48, -0.39, and -0.45, respectively, all P <0.01). CEC loss 1-12 months after surgery was significantly correlated with IL-1b, IL-7, IP-10 and RANTES concentrations (-0.46, -0.52, -0.48, and -0.43, respectively). Multiple regression analysis showed that IL-7 concentration was significantly associated with CEC loss 1-6 months after surgery (b = -0.65, P < 0.01) and IP-10 concentration was associated with CEC loss 1-12 months after surgery (β = -0.38, P < 0.05). These results suggest that not only inflammatory cytokines but also IL-7, a cytokine related to lymphocytes, may be involved in the depletion of CECs after DSAEK, particularly depletion that occurs relatively early.

Anti-inflammatory effects of acacia and guar gum in 5-amino salicylic acid formulations in experimental colitis

Findings from recent studies revealed a significant anti-inflammatory effect of polysaccharide-based excipients when formulated with classical drugs in experimental inflammatory bowel disease models. In this study, acacia and guar gum were investigated beyond their typical functionality for a possible additive anti-inflammatory effect when administered with 5-amino salicylic acid (5ASA) in murine experimental colitis. Anti-inflammatory effects of acacia and guar gum-based aqueous suspensions of 5ASA were evaluated in a murine experimental colitis. Acacia or guar gum (30 or 300 mg/kg) were administered via rectal administration alone or in combination with 5ASA (30 mg/kg). Disease activity, myeloperoxidase activity (MPO) and intratissue concentrations of various cytokines were assessed. Both acacia and guar gum separately showed significant effects in reducing the inflammatory markers in murine colitis model in vivo. When combined with the anti-inflammatory drug 5ASA, acacia showed a stronger therapeutic effect than guar gum, especially at the higher dose of acacia (300 mg/kg) which significantly reduced the inflammation in vivo compared to 5ASA alone (MPO, 5ASA: 5743 ± 1334, 5ASA + 30 mg/kg acacia: 3762 ± 2342; 5ASA + 30 mg/kg guar gum: 7373 ± 2115, 5ASA + 300 mg/kg acacia: 3131 ± 1012, 5ASA + 300 mg/kg guar gum: 6358 ± 2379; all U/g tissue). Acacia and guar gum separately showed significant anti-inflammatory effects in murine colitis, and furthermore, high dose acacia led to an additional therapeutic benefit when co-administered with 5ASA. These results indicate that further investigations are surely warranted in the search of better colitis therapy.

Influence of Plant Growth Regulators on Callogenesis and the Biomass of Cell Suspensions in Lily (Lilium ledebourii and Lilium regal)

Introduction: Lily is often described as one of the most widespread, commercial crops in the floriculture industry. Commercially grown cultivars are mostly propagated by bulb scales which it cost effective and uniformity in tissue culture conditions. Plant tissue culture techniques can effectively provide far-reaching implications in micro-propagation. Materials and Methods: This study was conducted to investigate the effects of various concentrations of cytokines and auxins on callogenesis and Biomass under in vitro conditions. In this experiment, the evaluations were aimed at measuring different characteristics in two lily cultivars, namely, Lilium ledebourii and Lilium regal. Results: The results showed significant values in all of the measured characteristics. The highest percentage of callogenesis was caused by 2 µm PIC plus 1 µm KIN in L. ledebourii (88.66%) and L. regal (88.66%). Also, the callus weight in both cultivars was obtained by applying the same combination of treatments. In the second experiment, the highest fresh biomass of the cell suspension occurred by applying 2 µm PIC plus 4 µm KIN. The maximum amount of fresh biomass in L. ledebourii (88.93 g/L) and L. regal (41 g/L) occurred on the 24th and 20th day of the culture, respectively. Conclusions: An efficient and fragile callus induction was developed by NAA, which nonetheless made the condition more suitable for cell suspension culture. These lily cultivars need high amounts of PIC as auxin to grow well in cell suspensions. By increasing the PIC level, the biomass accumulates more. These results can moderately optimize large-scale production of both fragile calli and cell suspension biomass.

Advanced-platelet-rich fibrin extract promotes adipogenic and osteogenic differentiation of human adipose-derived stem cells in a dose-dependent manner in vitro

Advanced platelet-rich fibrin (A-PRF) is an autogenous biological material obtained from peripheral blood. A-PRF extract (A-PRFe) contains a high concentration of various cytokines that are increasingly appreciated for their roles in improving stem cell repairing function during tissue regeneration. However, the optimal A-PRFe concentration to stimulate stem cells is unknown. This study aimed to identify the optimal concentrations of A-PRFe to promote adipogenic and osteogenic differentiation of human adipose-derived stem cells (ASCs). We produced A-PRFe from A-PRF clots by centrifuging fresh peripheral blood samples and isolated and identified ASCs using surface CD markers and multilineage differentiation potential. Enzyme-linked immunosorbent assay (ELISA) showed the concentrations of several cytokines, including b-FGF, PDGF-BB, and others, increased gradually, peaked on day 7 and then decreased. Cell proliferation assays showed A-PRFe significantly stimulated ASC proliferation, and proliferation significantly increased at higher A-PRFe doses. The degree of adipogenic and osteogenic differentiation increased at higher A-PRFe concentrations in the culture medium, as determined by oil red O and alizarin red staining. Reverse transcription polymerase chain reaction (RT-PCR) showed that expression levels of genes related to adipogenic/osteogenic differentiation (PPARγ2, C/EBPα, FABP4, Adiponectin, and ALP, OPN, OCN, RUNX2), paracrine (HIF-1α, VEGF, IGF-2) and immunoregulation (HSP70, IL-8) function were higher in groups with a higher concentration of A-PRFe than in lower concentration groups. This study demonstrates that A-PRFe is ideal for use in ASC applications in regenerative medicine because it improves biological functions, including proliferation, adipogenic/osteogenic differentiation, and paracrine function in a dose-dependent manner.

Increased cytokine expression in the choroid plexus stroma and epithelium in response to endotoxin-induced systemic inflammation in mice.

Sepsis-associated encephalopathy (SAE) is characterized as diffuse brain dysfunction in patients with excessive systemic inflammatory reaction to an infection. In our previous studies using a mouse model of SAE with intraperitoneal injection of lipopolysaccharide (LPS), tissue concentrations of various cytokines were elevated in the entire brain parenchyma 4 and 24 h following LPS administration. Cytokines elevated at 4 h were produced by the choroid plexus, leptomeninges and vascular endothelium, while those at 24 h were produced by astrocytes. Interleukin (IL)-1β did not increase in the concentration in the brain parenchyma during the period from 1 to 24 h following LPS. In the present study, we hypothesized that the intracranial cells that initially respond to systemic inflammation are situated in the choroid plexus and produce IL-1β to initiate cytokine-mediated reactions. We quantified the transcript levels of related cytokines within the choroid plexus and specified the choroid plexus cells that are involved in the immediate cytokine-mediated responses. Mice received LPS or saline by intraperitoneal injection. Four hours after treatments, the choroid plexuses were isolated and subjected to cytokine gene expression analyses using real-time reverse transcription-polymerase chain reaction. Another group of mice was fixed at 1, 4 and 24 h after treatments and the expression of cytokines and receptors was studied with double immunohistofluorescence staining. The transcript levels of IL-1β, CC-motif ligand (CCL)2, CXC-motif ligand (CXCL)1, CXCL2 and IL-6 in the choroid plexus were significantly increased in mice treated with LPS compared to saline control. The IL-1β expression was remarkable in choroid plexus macrophages at 1 and 4 h but not in the brain parenchyma. Choroid plexus stromal cells expressed IL-1 receptor type 1 (IL-1R1). The IL-1R1-bearing stromal cells produced CCL2, CXCL1, CXCL2 and IL-6 at 4 h. Choroid plexus epithelial cells expressed CXCR2, a common receptor for CXCL1 and CXCL2. Choroid plexus epithelial cells also expressed CCL2, CXCL1 and CXCL2 at 4 h, and IL-1R1-bearing stromal cells expressed CXCR2. Therefore, in response to systemic LPS injection, one of the intracranial reactions was initiated within the choroid plexus using IL-1β derived from macrophages. The choroid plexus stromal cells subsequently had elevated expression of CCL2, CXCL1, CXCL2 and IL-6. The choroid plexus epithelial cells also had elevated expression of CCL2, CXCL1 and CXCL2. The presence of receptors for these cytokines on both epithelial and stromal cells raised the possibility of reciprocal interactions between these cells. The results suggested that the immediate early responses exerted by the choroid plexus are relevant to understanding how SAE is initiated in clinical settings.

Cytokine status in the omentum and peritoneum in metastatic gastric cancer.

e16557 Background: Gastric cancer (GC) is one of the most common malignant tumors and the second most common cause of cancer deaths in Russia. Many researchers note the ambiguous role of cytokines in the pathogenesis of tumor processes. The purpose of the study was to determine characteristics of the cytokine status in the omentum and peritoneum of patients with metastatic GC. Methods: The main group (Mgr) included 23 patients with T2-4N0-3M1 GC with metastases to the peritoneum and greater omentum, the mean age 58.9±9.7 years. The comparison group (Cgr) included 20 non-cancer patients, the mean age 55.9±9.7 years. Tissues of the omentum (OT) and peritoneum (PT) were obtained from all patients during cytoreductive and diagnostic surgeries. Ex tempore tissues were disintegrated using BD Medimachine (USA) with 0.9% NaCl; the cell suspension was centrifuged, and the supernatant was collected and aliquoted. Levels of cytokines IL-1β, IL-2, IL-6, IL-8, IL-18, TNF-α, IL-10 (Vektor-Best, Russia) and IL-1RA (eBioscience, Austria) were determined by ELISA. The results were expressed as the specific content per 1 g of protein evaluated by the biuret method (pg/mL/g of protein). Statisical analysis of the results was performed with the Statistica 10 program (StatSoft Inc., USA). Significance of differences was evaluated using the Mann-Whitney and Wilkinson tests; differences were considered significant at p &lt; 0.05. Results: In Cgr, levels of IL-6, IL-8, IL-18 and IL-1β in OT were 4.1±1.1, 3.4±0.7, 21.2±5.5 and 10.8±2.1, while in Mgr – 55.3±24.4, 32.5±13.8, 144.9±48.4 and 21.01±5.4 pg/mL/g of protein, i.e. 13.7, 9.5, 6.9 and 1.9 times (p≤0.05) higher than in Cgr, respectively. The specific content of IL-1RA in OT was 12.6 times higher, p≤0.05 (215.7±69.8 vs. 2723.1±730.1), and the IL-10 concentration did not differ significantly. In PT of Cgr, levels of IL-8, IL-18 and IL-6 were 9.8±7.3, 35.2±21.7, 7.5±3.7, and in Mgr – 250.7±21.4, 322.9±16.9, 61.1±27.6 pg/mL/g, i.e. 25.7, 9.2 and 8.2 times (p≤0.05) higher than in Cgr. IL-1RA in PT of Mgr was 24.4 times higher than in Cgr, p≤0.05 (7656.2±3160.3 vs. 357.69±180.86). Conclusions: A certain microenvironment is formed in OT and PT affected by metastases, with specific features in the ratio and concentrations of various cytokines, which undoubtedly contribute to the formation of a secondary tumor.

Treatment of Auricular Hematomas by OK-432: How and Why It Works.

The aim of this article was to investigate the effectiveness and underlying mechanisms of OK-432 therapy in patients with auricular hematomas. Case series with planned data collection. We tried this therapy in 47 patients with auricular hematoma between April 2008 and August 2018. We aspirated as much of the fluid content of each lesion as possible with a 21-gage needle. We injected OK-432 solution into the lesion with the same needle that we used for aspiration. We performed this treatment at an outpatient basis without hospitalization. Disappearance and marked reduction of the lesion were observed in all patients who had this therapy, and local scarring and deformity of the auricle did not occur in any patients. As adverse effects, local pain at the injection site and fever (37-38.5°C) were observed in some cases of the patients who had this therapy. The concentrations of various cytokines in each aspirate before and after OK-432 therapy were investigated. The production of tumor necrosis factor-α, interleukin-6, interleukin-8, interferon gamma, vascular endothelial growth factor, and periostin was significantly elevated in the aspirate fluid after OK-432 therapy. OK-432 therapy is simple, easy, safe, effective, and can be used as a substitute for surgery in the treatment of auricular hematoma.

Interaction Between Regulatory T Cells and Antibody-Producing B Cells for Immune Responses at the Upper Respiratory Mucosa Against Nontypeable Haemophilus influenzae: In Vitro Assay Model.

The aim of this study was to investigate the effect of regulatory T cells (Tregs) on B-cell immune responses against outer membrane protein (OMP) from nontypeable Haemophilus influenzae (NTHi) in vitro, to clarify its exact mechanism from an immunologic standpoint. Mice were vaccinated intranasally with OMP to induce OMP-specific immune responses in the nasal mucosa. Mononuclear cells (MNCs) were collected from the nasal mucosa, and Tregs and helper T (Th) cells were isolated separately from the spleens of those mice. Three different cell culture groups were allocated: MNCs cocultured with Tregs, MNCs cocultured with Th cells, and MNCs cultured alone. At 24 and 72 hours after cell culture, the concentrations of various cytokines and antibodies in culture supernatants were measured to assess the effects of Tregs and Th cells on B-cell responses. Cytokine levels and specific anti-OMP antibody levels in culture media were determined using enzyme-linked immunosorbent assay. CD69 or CD80 expression on B220-positive cells was detected using flow cytometric analysis. Th1 and Th2 cytokine concentrations were significantly elevated in the 3 groups incubated with OMP from 24 to 72 hours. Additionally, interleukin-10 levels were significantly higher in the Treg and Th groups than in the control group. Levels of OMP-specific immunoglobulin A did not differ significantly among the groups. The ratios of CD69+B220+ B2 cells were nearly the same in the 3 groups; however, the ratio of CD80+B220+ B2 cells was higher in the control group than in the Treg and Th groups during incubation. Tregs and Th cells did not affect OMP-specific immunoglobulin A production in this study. However, these cells may partially inhibit B-cell functions, such as T-cell activation. These inhibitory effects may be related to interleukin-10.

In vitro cytokine synthesis in unstimulated and mitogen-stimulated peripheral blood mononuclear cells from individuals with schizophrenia

Increasing evidence has shown that the immune system is involved in the schizophrenia development, with alterations in immune cell reactivity being one possible factor contributing to its pathogenesis. The purpose...

Characterization of two potential immunotherapeutics in tumor infiltrating lymphocytes (TILs) using flow cytometry and Meso Scale Discovery.

e15052 Background: Inter-K Peptide Therapeutics has developed two synthetic compounds that modulate kinase activity. To investigate the potential of these compounds as cancer immunotherapeutics, a flow cytometric intracellular cytokine staining (ICS) assay was developed to investigate the effect of the compounds on immune cells isolated from tumors from subjects with non-small cell lung cancer (NSCLC). In addition, the concentration of various cytokines was measured in cell culture supernatant after treatment with the compounds. Methods: After optimizing the assays in PBMC from healthy donors, the assay was used for clinical specimens. Tumor infiltrating lymphocytes (TILs) were isolated from two resected tumors by mechanical disaggregation. TILs were stimulated with or without anti-CD3/CD28 antibodies with and without Inter-K Compound A or Compound B for 72 hours. Following stimulation, cell culture supernatants were analyzed for 10 proinflammatory-related cytokines using the Meso Scale Discovery (MSD) assay. TILs were analyzed by flow cytometry with a 10-color immunophenotyping panel to measure the expression of functional markers CD25, IL-2, IFNg and Ki67 in NK, NKT, CD4 T and CD8 T cells. Results: Immunomodulation induced by Compound A treatment included an over two-fold increase in CD25 expression in CD4 T cells, statistically significant increases in IL-6 and IL-8 production while a decrease in Ki67 expression in CD8 T cells and a reduction in IL-2, IFNg, TNFa, IL-4, IL-10 and IL-13 production was observed. Compound B did not appear to have immunomodulatory properties. Conclusions: This work provides an example of the utility of flow cytometry in compound evaluation. In this case, preliminary data suggests that Compound A but not B was able to modulate the response in TIL samples in vitro, and supports the continued investigation of Compound A as an immunotherapeutic. Although TIL specimens are challenging to work with due to tumor availability and low cellular recovery, as the target for the immunotherapy, it is important that compound characterization be conducted in TIL in addition to healthy donor PBMC and cell lines.

Acute adrenal cortex injury during cardiopulmonary bypass in a canine model

ObjectiveCardiopulmonary bypass (CPB) might induce systemic inflammatory responses that cause acute injuries to multiple organs. However, no direct evidence exists to determine whether CPB leads to adrenal cortex injury or to describe its underlying mechanism. MethodsTwelve healthy adult beagles were randomly assigned into control and CPB groups. After cannulation, mild hypothermia CPB was performed in the CPB group but not in the control group. The serum concentrations of various cytokines, cortisol, and aldosterone were assessed. Adrenal cortex injuries were evaluated using standard histological methods. Steroidogenic enzymes and the nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome pathway were detected using quantitative polymerase chain reaction and Western blot analysis. ResultsDuring CPB, serum interleukin (IL)-6, IL-8, IL-10, tumor necrosis factor α, cortisol, and aldosterone levels were significantly higher in the CPB group. The pathologic study revealed higher injury scores (3.6 ± 0.6 vs 0.7 ± 0.7) and significantly more severe edema, inflammatory cell infiltration (lymphocytes and neutrophils), and apoptosis in the CPB group. The electron microscopic examination showed swollen mitochondria, ruptured mitochondrial cristae, reduced lipid droplets, and increased secondary lysosomes in the CPB group. The mRNA expression levels of NLRP3 and the protein levels of 17α-hydroxylase and IL-1β in adrenal tissue were significantly upregulated in the CPB group. ConclusionsCPB induces significant systemic and local inflammation in the adrenal cortex and results in cytological architectural and ultrastructural alterations in adrenocorticocytes. In addition, the NLRP3 inflammasome pathway might promote adrenal gland injury during CPB and might represent a novel potential therapeutic target.

Changes in aqueous concentrations of various cytokines after intravitreal bevacizumab and subtenon triamcinolone injection for diabetic macular edema.

The purpose of this study was to compare the changes in the aqueous cytokine levels after intravitreal bevacizumab with those after combined intravitreal bevacizumab and subtenon triamcinolone injection in diabetic macular edema (DME). This study examined 24 eyes of 23 patients with DME. Each patient with DME received randomly either an intravitreal injection of bevacizumab (IVBe) or IVBe with a subtenon triamcinolone injection (IVBe+STTA). Best corrected visual acuity and foveal thickness were evaluated and aqueous samples were obtained before and 4weeks after the injection. The aqueous concentrations of interleukin (IL)-6, IL-8, interferon-induced protein (IP)-10, monocyte chemotactic protein (MCP)-1, platelet-derived growth factor (PDGF)-AA, and vascular endothelial growth factor (VEGF) were measured using a multiplex bead assay. After the injection, the foveal thickness decreased more in the IVBe+STTA group than in the IVBe group (P=0.042). The MCP-1, PDGF-AA, and VEGF levels decreased significantly in the IVBe+STTA group (p=0.013, p=0.004 and p=0.018 respectively), but only the VEGF level decreased in the IVBe group (p=0.001). IL-8 was significantly increased in the IVBe+STTA group (p=0.003) but the changes in the VEGF levels were smaller than in the IVBe group (p=0.025). Intravitreal bevacizumab and subtenon triamcinolone injection reduces the VEGF, MCP-1 and PDGF-AA levels and increases the IL-8 level in the plural cytokine profiles of patients with DME, which might explain the limited therapeutic effect of combination therapy.